CN101942413B - Birth defect cell bank and construction method thereof - Google Patents

Abstract

The invention relates to a birth defect cell bank for use in the field of medicines and a construction method thereof. The construction method is mainly characterized by comprising: constructing recombinants SV40t/pcDNA3.1 by using a BamH I, SV40 DNA and T4 DNA ligase and gene vectors pcDNA3.1; amplifying and purifying by competent Escherichia coli; importing the recombinants into in-vitro suspension birth defect cells, which are digested by 0.01 percent collagenase II, or anchorage-dependent birth defect cells, which are about to enter or just enter an exponential phase, contain 90 percent spindle cells and have a rendezvous rate of 55 to 65 percent in culture solution containing 20 percent of fetal calf serum and insulin at a concentration of 5 to 10 mol/L, by lipofectin transfection, and integrating the recombinants with the DNAs of the cells; using cells which contain positive recombinants and are screened by G418 and cells which are transfected with EB viruses as passage cells, performing amplified culture, constructing a birth defect cell line, and freezing and storing the cell line in liquid nitrogen; and naming the cell line by Birth Defect or by using the first letter of the English name of the virus type and marking related data information for centralized storage of the birth defect cell resources. Thus, the problems of lack of scientific research materials and lack of specific birth defect cell banks are solved.

Description

Birth defect cell bank and construction process thereof

The present invention relates to birth defect cell bank and construction process thereof, be mainly used in the concentrating of inborn defect resource in medical research field, persistence provide scientific research material for the research of birth defect disease.

Inborn defect refers to that fetus is already present structure or dysfunction in utero.Account for natus 4-6%, reach kind more than 10,000, wherein modal have congenital heart disease, mongolism (Down's syndrome, trisomy 21 syndromes), anencephalus, harelip, deaf and dumb etc.

People's Republic of China's [President makes (1994) No. 33] makes " set up the Ministry of Health and antenatal diagnosis center, each province and city, from June 1 nineteen ninety-five, implement national inborn defect Prenatal Screening and antenatal diagnosis ".After this, also determine that annual September 12 is for " China's prevention inborn defect day ", all kinds of research topics such as " key special subjects or preferential themes " that the intervention of inborn defect and the research of diagnostic methodology thereof are put into, country has dropped into a large amount of manpower and materials, but the progress of not yet making a breakthrough property so far, its diagnostic method almost also rests on level originally.As topmost methods for prenatal diagnosis also continue to use the seventies in last century just ongoing, working efficiency very low, 0.5-1% pregnant woman can be caused miscarry have the property created amniocentesis chromosome analysis technology; The Prenatal Screening result false negative rate of the inborn defect (21-Trisomy) that wherein hazardness is maximum is 0.05% (colony's overall incidence is 0.1-0.15%), and namely rate of missed diagnosis is up to 35%; And its false positive rate is up to 99%, but also the birth of the mongolism infant of about 65% can only be prevented.So the technological lag of current Prenatal Screening and diagnosis, far can not meet the requirement of antenatal diagnosis.

Trace it to its cause, mainly also there is no a kind of good method energy centralized collection and persistence inborn defect sample at present, cause its research material poor.Suffer from embryo or the fetus of Severe birth defect, abandoned voluntarily by pregnant woman after death in uterus often or abandon after doctor's process.

In other medical research field, in order to ensure more sufficient Scientific Research Resource, in recent years, existing increasing Chinese scholars and academic institution are devoted to the foundation in various types of cells storehouse, save a large amount of normal or have the cellular resources of pathological characters with this.Various types of cell bank has all been had in the U.S., Britain and Japan etc.As built in the U.S. " standard cell lines storehouse (ATCC) ", " human inheritance's mutant cell storehouse (HGMR) ", " cell aging cell bank (CAR) " etc., Britain's built " embryonic stem cell bank " etc., Japan built " deciduous teeth stem-cell research storehouse ", " induction versatile stem cell " etc., China has built " Chinese important medical biotechnology resource preservation storehouse ", " Chinese different nationalities Immortalized Cell Bank ", " Chinese mankind's inheritance resources platform ", " people's mandibular condylar chondrocytes storehouse ", " human placenta's stem cell bank ", " Human keratinocytes storehouse ", " umbilical cord mesenchyma stem cell ", " schizophrenia Immortalized Cell Bank ", " T cell storehouse ", " cord blood stem cell bank ", " hemopoietic stem cell bank ", " people's embryo adenopituicyte storehouse ", " tumour cell storehouse ", " immortal lymphoblastoid cell bank of keloids pedigree ", " immortal lymphoblastoid cell lines of long-lived people " etc.

Different cells is saved separately in the cell bank that these are different.As " human lung adenocarcinoma cell, human T lymphocyte leukemia cell, wild type human C-KIT recipient cell strain (category-B), people's glioblastoma cells, Humanmachine tumour, human lung adenocarcinoma cell, the strain of human lung adenocarcinoma taxol resistance, drug-resistant cell strain, people's band cancer cells, Humanmachine tumour, Human Prostate Cancer Cells, Proliferation of Human Ovarian Cell, human renal carcinoma cell, the dual-gene transfection cell strain of people APP-PS1 (HEK293), human chorionic tumour cell, CNE1-1mp ' 1 people transgenosis Nasopharyngeal Carcinoma Cell Line, CNE1-1mp-NF-KB people's transgenosis Nasopharyngeal Carcinoma Cell Line, CW-2 human colon adenocarcinoma cell, Daudi human B lymphocyte knurl, DMF7 dibit point HC-KIT recipient cell strain (category-B), F2-2 people 5-8 transgenosis Nasopharyngeal Carcinoma Cell Line, C33, ASP2 human embryonic kidney cell transformant, FGC Human Prostate Cancer Cells, FG lefteye flounder cell, F56 Human adenocarcinoma cells, Gpc-1 Human Prostate Cancer Cells, GLC-82 human lung adenocarcinoma cell, GT1.1 people's pituitary tumor cell, GBC-SD Human gallbladder carcinoma cell, HK-2C human embryonic kidney epithelial cells, HK-2 people's renal cells, H9 human T lymphocyte, H292 people's epidermis lung carcinoma cell, HB human oral cancer cell, HBE human bronchial epithelial cell, BL-100 human breast cell, HBZY-1 people's renal glomerulus pith mother cells system, HLF-02 human embryonic lung diploid fibroblast, HUVEC Human umbilical vein endothelial cells, HaCat people's immortalization epidermic cell, HuT 78 human T cell leukemia cell, Hurt-78 HTL, HaCAT people's immortalization epidermic cell, JAR human placenia cancer cells, Jurket77 human T lymphoma Cell subline, LCC9 human breast carcinoma estrogen antagonist persister, LCC2 human breast carcinoma Tamoxifen persister, 10104 human lymphoma cells (category-B), the dual-gene transfection cell strain of 7WCY1.0 people APP-PSI (CHO) (category-B), 7WD10 people's app gene transfection cell strain (CHO) (category-B), people-PS1 (M146L) dual-gene transfection cell strain (CHO) (category-B), the dual-gene transfection cell strain of 7WPS1 people APP-PS1 (CHO) (category-B), the low transfer lung carcinoma cell of 95-C people, 95-D people's height transfer lung carcinoma cell, 973 human lung adenocarcinoma cells, 5637 human bladder cancer cells, 8910PM adenocarcinoma ovaries, T-CEM human T cell leukemia cell etc.

These different cells preserved in different cell banks or clone, because various types of cells is distinct, method and the condition of its structure clone are also different, if any Epstein-Barr virus revulsion, granzyme-induced method, papilloma virus revulsion, Adenovirus E1A gene revulsion, simian sarcoma virus (SV40) revulsion.But the cell in current most cell bank, it is all the feature according to Human peripheral blood B lymphocytes surface with Epstein-Barr virus acceptor, make the phase become immortalization bone-marrow-derived lymphocyte clone with ebv infection Human peripheral blood B lymphocytes, then frozenly in liquid nitrogen, be built into cell bank.The experimental technique of this Epstein-Barr virus structure bone-marrow-derived lymphocyte system and methods of sample collection are all more for convenience, obtain more general application, but be only confined to be all can not set up clone or cell bank with Epstein-Barr virus to there is no the cell of Epstein-Barr virus acceptor such as T lymphocyte, white corpuscle, histocyte etc. to there being the bone-marrow-derived lymphocyte of Epstein-Barr virus acceptor to build.As the stillborn foetus of inborn defect or the live histocyte of tire and the aging fetus cast-off cells come off in amniotic fluid just can not build immortalized cell line with Epstein-Barr virus and be stored in cell bank.

At present, there is not yet the bibliographical information building birth defect structure clone both at home and abroad with Epstein-Barr virus or simian sarcoma virus (SV40) etc., also there is not yet both at home and abroad about structure birth defect cell bank and in order to centralized collection, the histiocytic bibliographical information of persistence inborn defect.In order to solve the problem, propose the present invention.

The object of the invention is to provide to concentrate and preserve various inborn defect resource and can study for it birth defect cell bank and construction process thereof that scientific research material is provided.

The object of the present invention is achieved like this: with BamH I, SV40DNA, T4DNA ligase enzyme and genophore pcDNA3.1 build SV40T/pcDNA3.1 recon, increase through competence intestinal bacteria, purifying, the in vitro suspension cell that digests through 0.01% collagenase II is imported or containing 20% foetal calf serum with lipofection, be in the nutrient solution of 5 ~ 10nmol/L Regular Insulin and will enter or just enter logarithmic phase, spindle cell accounts for more than 90%, converge the adherent growth cell that rate reaches 55% ~ 65%, the DNA of recon and cell is integrated, that screens with G418 contains the cell of positive recombinant and the cell of Epstein-Barr virus transfection, go down to posterity, enlarged culturing, structure birth deficient cells system is also frozen in liquid nitrogen, name with inborn defect (Birth Defect) and sick first English alphabet of planting thereof and indicate related data information, build birth deficient cells system and cell bank.

The present invention imports inborn defect cell with the SV40 large T antigen gene of purifying through genophore pcDNA3.1 and builds clone, the tryptic digestion that wherein inborn defect histocyte need not be conventional with 0.01% collagenase II digestion, digested or make cell suspension with the collagen only making to cause cytoadherence, the protein decreasing cell walls is digested and injure cell, makes the success ratio of cell cultures bring up to more than 95% by general about 85%; Select the defined medium containing 20mL/L foetal calf serum, 5 ~ 10nmol/L Regular Insulin, make cell can not grow too fast and affect the integration of SV40 large T antigen gene, also can not make because lacking nutrition or Growth of Cells stimulating factor cell do not meet the requirements of converge rate, do not enter logarithmic phase before with regard to premature death, or logarithmic phase shortens; Clone recovery cultivation after frozen 1 month in-196 DEG C of liquid nitrogen of preparation, all can grow attached cell; When making clone Chromosome Identification, the consumption of colchicine and action time are conventional 5 ~ 10 times, and chromosome separation is increased mutually, are enough to counting and analyze.The inborn defect clone built up thus and cell bank thereof, concentrate the viable cell resource saving inborn defect, research for all kinds of inborn defect provides scientific research material, for the research, the improvement and bring new ideas that accelerate birth defect diagnosis method lay the foundation, and solve at present that inborn defect scientific research material is poor and without the problem of single-minded birth defect cell bank both at home and abroad.

The following detailed description of realizing implementation method of the present invention:

1, the extraction of SV40 large T antigen DNA: 1. SV40DNA enzyme is cut: the SV40 freeze dried powder or the SV40 plasmid that contain large T antigen gene from commercially available purchase, be dissolved in appropriate H ₂in O or TE damping fluid, add 2uL10 × enzyme cutting buffering liquid and 18uL H ₂o, adds restriction enzyme BamH I (1-5U/ugDNA), 37 DEG C of incubation 1h, 75 DEG C of heating 15min, and inactivator, adds 5uL electrophoresis sample loading buffer (also by adding 0.5mol/L EDTA) termination reaction in order to electrophoresis.2. SV40DNA electrophoresis: power taking swimming level agarose is made into 10% sepharose with electrophoretic buffer, pour on the gel casting platform sealed, plug sample comb, from glue platform removing envelope band after gelling is solid, extract comb, put into the electrophoresis chamber being added with enough electrophoretic buffers, damping fluid exceeds gel surface and is about 1mm, DNA sample is prepared with 10 appropriate × sample loading buffer, then with pipettor, sample is added in sample well, and do suitable DNA molecular amount standard control thing simultaneously, connect electrode, DNA anode is moved, when under the voltage of 1-10V/cm gel, electrophoresis is to the distance of enough isolation of DNA fragments, powered-down.3. from agarose, about 2600bp SV40 large T antigen DNA is separated: under 300-360nm long wave ultraviolet light source, (use long wave ultraviolet light source to prevent DNA damage) cut in loading dialysis tubing by the gel-tape containing target DNA fragments, 2ml electrophoretic buffer is added in dialysis tubing, make it submergence gel, and emptying steam bubble, dialysis tubing level is put into electrophoresis chamber (length direction is parallel with electrophoresis), add appropriate amount of buffer solution by dialysis tubing submergence (about 6-7mm), switch on power, 150 volts of electricity are washed, observe under ultraviolet lamp and treat that DNA all shifts out gel, change direction of an electric field and continue energising 1 minute, from dialysis tubing, sucking-off damping fluid is in 1-5ml Eppendorf pipe, add 1.5 times of volume propyl carbinols, EB is removed in mixing extracting, on desk centrifuge 2 minutes the most at a high speed, suck upper strata butanol solution, repetition secondary like this, equal-volume phenol chloroform (2) extracting 2 times is added in the solution of lower floor speech DNA, supernatant proceeds in another Eppendorf pipe and adds 1/10 times of volume 3M NaAc, 2 times of volume precooling dehydrated alcohols, spend the night in 20 DEG C, 12000g, at 4 DEG C centrifugal 10 minutes, obtain DNA precipitation, abandon supernatant, dry ethanol is abandoned after adding 70% washing with alcohol 2 times, add 50 μ l TE dissolving DNAs.In addition, also target DNA fragment is separated, is purified by available low melting-point agarose gel method, DNA filter membrane inserted sheet method etc. from gel.

2, the connection of SV40 large T antigen DNA and pcDNA3.1 genophore: get the above-mentioned DNA composition of 9 μ l (0.1-5 μ g), 10 μ l 2 × connect damping fluid, 1 μ l 10mmol/L ATP, T4DNA ligase enzyme (20 ~ 500 sticky end unit) or e. coli dna ligase, pcDNA3.1 empty carrier mix, 15 DEG C of incubation 24h, are built into SV40T/pcDNA3.1 recon.

3, the amplification of SV40T/pcDNA3.1 recon, Isolation and ldentification: the 1. preparation of E. coli competent: its basic skills is ice-cold CaCl ₂or the process such as multiple divalent positively charged ion bacterium, making it to enter competence is transformed, and uses CaCl ₂prepare fresh or freezing competent escherichia coli cell, be usually used in preparation in quantity competence bacterium, this law is applicable to most of coli strain, operating process is summarized as follows: cultivate picking single bacterium colony (as bacillus coli DH 5 2) the fresh plate of 16 ~ 20h from 37 DEG C, or 16 ~ 20h overnight culture that 1ml is fresh, forward one to containing in 1L or the 500ml flask of 100mlLB substratum, about 2 ~ 3h (rotary shaker 200 ~ 300r/min) is cultivated in 37 DEG C of violent joltings, OD600 value ≈ 0.4 is measured every 20 ~ 30min, aseptically bacterium is transferred to one, in ice-cold 50ml polypropylene centrifuge tube, 10 ~ 20min is placed on ice, in 4 DEG C with SorvallGS2 rotary head (or the rotary head matched with its centrifuge tube) with the centrifugal 10min of 4000r/min, to reclaim cell, nutrient solution reciprocal, pipe is inverted 1min to flow to end to make the trace nutrient solution of final residual, with the ice-cold 0.1mMCaCl of 10ml ₂resuspended every part of precipitation, be put on ice, in 4 DEG C with SorvallGS3 rotary head (or its corresponding rotary head) with the centrifugal 10min of 4000r/min, to reclaim cell, pour out nutrient solution, pipe is inverted 1min to flow to end to make the trace nutrient solution of final residual, the 0.1M CaCl of every 50ml initial incubation thing 2ml ice precooling ₂resuspended every part is sunk calmly, now, rapidly cell can be distributed into aliquot, freezing in liquid nitrogen,-70 DEG C of storages are for subsequent use, from every kind of competent cell suspension, getting 200 μ l with the sterile pipette tip of cooling transfers in aseptic Eppendorf tube, often pipe adds DNA or ligation mixture (volume≤10 μ l, DNA≤50ng), rotate gently to mix content, 30min is placed in ice, centrifuge tube is put into pre-heating on the test-tube stand in the circulator bath of 40 DEG C, place 90s ~ 2min, do not shake test tube, fast pipe is transferred in ice bath, cell is made to cool 1 ~ 2min, every centrifuge tube adds 800 μ lSOC substratum, with water-bath, substratum is warmed to 37 DEG C, then pipe is transferred on 37 DEG C of shaking tables, incubation 45min makes bacteria resuscitation, and antibiotic-resistance marker's gene of expression plasmid coding, the competent cell that proper volume (each 90mm flat board can reach 200 μ l) has transformed is transferred to containing on 200mmol/LMgSO4 and corresponding antibiotic SOB substratum, flat board is placed in room temperature to be absorbed to liquid, be inverted plate, in 37 DEG C of cultivations, bacterium colony can be there is after 12 ~ 16h.2. the screening of recon, amplification and extraction: select single colony inoculation in the aseptic LB substratum of 5mL or rich medium (as super broth or TB super broth substratum) with sterile toothpick or disinfection inoculation pin, after overnight incubation, join 500mL again containing in the 2L flask of LB substratum (containing suitable microbiotic), then be cultured to state of saturation (OD in 37 DEG C ₆₀₀≈ 4, for improving output, the flask of the comparatively large and band traverse baffle of surface-area should be adopted to increase venting quality as far as possible, jolting speed should be greater than 400r/min), in 4 DEG C, the centrifugal 10min of 6000g, by the resuspended precipitation of 4mL GTL solution, and transfer to (bacterial precipitation can be preserved at-20 DEG C or-70 DEG C of indefinitely) in the high speed centrifugation pipe of a volume>=20mL, add the GTE solution containing 25mg/mL N,O-Diacetylmuramidase that 1mL newly joins, resuspended precipitation, 10min is placed in room temperature, add 10mL and newly join NaOH/SDS solution, and mixing is until liquid becomes homogeneous gently, limpid and thickness, in placing 10min on ice, add 7.5mL acetic acid solution, stir until viscosity declines and forms large precipitation gently with suction pipe, in placing 10min on ice, in 4 DEG C, the centrifugal 10min of 20000g, supernatant is poured into gently in another clean centrifuge tube, if there is visible drift can by several layers of filtered through gauze, add the Virahol of 0.6 times of volume, put upside down mixing, room temperature places 5 ~ 10min, in room temperature, the centrifugal 10min of 1500g, add the washing precipitation gently of 2mL 70% ethanol, then of short duration centrifugal fast, suck ethanol, and vacuum-drying (precipitation can be preserved for a long time at 4 DEG C).3. the qualification of recon: the above-mentioned DNA (containing recon SV40T/pcDNA3.1) extracted from competence intestinal bacteria, the same method restriction enzyme BamH I carries out enzyme and cuts, 10g/L agarose gel electrophoresis is identified, obtain 2 bands that size is about 2600bp and 5600bp, the former meets the size of SV40T fragment in GenBank.

4, SV40T/pcDNA3.1 recon imports screening and the amplification of inborn defect cell and positive colony thereof: the 1. collection of inborn defect cell, process and preculture: with aseptic technique be extracted in birth defect disease diagnose in the lymphocyte of surplus, fetus amniotic fluid cast-off cells, wherein histocyte stroke-physiological saline solution rinsing several, then be cut into close to individual cells, or freezing at low temperatures rear grinding, about 10 minutes are digested with 37 DEG C of physiological saline containing 0.01% collagenase II, make into individual cells, centrifugally remove supernatant liquor, with stroke-physiological saline solution or cell culture medium suspension cell, centrifugally again remove supernatant liquor, after cleaning 2 to 3 times, 1 × 10 is about with final concentration of cells ⁵/ mL is for subsequent use, the cell got above-mentioned cell in single dispersion or become single dispersion is after treatment inoculated in containing 5 ~ 10nmol/L Regular Insulin, in the RPMI1640 liquid of 20% foetal calf serum or be inoculated in containing 20% foetal calf serum, in the low sugar DMEM cell culture medium of 5 ~ 10nmol/L Regular Insulin, be placed in 37 DEG C, in volume fraction 5%CO2 incubator, cell attachment is cultivated about 3 ~ 4 days, cellular form is fusiformis, little circular cell is less than 10%, the rate of converging of attached cell reaches 75% ~ 85%, be in when just entering logarithmic phase, namely consider to collect culturing cell, first blot the nutrient solution in clean culturing bottle, add the collagenase II liquid (can cover at the bottom of whole bottle with Digestive system and be as the criterion) of 1-2ml0.01%, leave standstill 2-10min (under microscope dynamic monitoring), suck collagenase II liquid, add low sugar DMEM or RPMI1640 nutrient solution, culture in glassware liquid is drawn with suction pipe, repeatedly blow and beat bottle parietal cell, form cell suspension, proceed to centrifuge tube centrifugation, remove supernatant liquor, after cell precipitation cleans 2 times with above-mentioned nutrient solution, make suspension, import as recon.2. the importing of SV40T/pcDNA3.1: method 1: in above-mentioned inborn defect cell, select lipofection, by above-mentioned 2 × 10 ⁵individual cell is inoculated in 35mm culture dish, at 37 DEG C of CO ₂24 ~ 36h cultivated by incubator, make Hemapoiesis individual layer, cellular form be fusiformis, there is not yet or be rarely seenly less than 10% little circular cell, the rate of converging of attached cell reaches 55% ~ 65%, is in when entering or just entered logarithmic phase, namely transfection recon SV40T/pcDNA3.1 is considered, in 1.5ml Eppendorf tube, prepare following solutions: pipe A, SV40T/pcDNA3.1 is dissolved in 100 μ l serum-free mediums; Pipe B, 20 μ l Lipofectamine are dissolved in 80 μ l serum-free mediums, mixed by pipe A and pipe B, the underlying 45min of room temperature, after sucking cell culture fluid, with serum-free medium washed cell 2 times, in Lipofectamine-SV40T/pcDNA3.1 mixture, add 1ml serum-free medium, mix gently, then drop in Tissue Culture Dish, then 1ml serum-free medium is added, at CO ₂10h cultivated by incubator, sucking-off transfection liquid, and add 4ml complete culture solution and continue to cultivate 16h, discard nutrient solution, changing concentration is 400mg ° of L ^-1g418 nutrient solution continue to cultivate, within 8 ~ 10 days, select individual cells colony to carry out subclone afterwards, strengthen G418 concentration after enlarged culturing again to 800mg ° of L ^-1, in the G418 environment of high density, amplification of going down to posterity can be carried out by the clone of stable growth, in addition after inborn defect cell and SV40 or Epstein-Barr virus co-cultivation, infect viral DNA and the inborn defect cell occurring to integrate carries out amplification of going down to posterity.Method 2: draw 1/10-1/40 cell suspension, being mixed with final concentration is 1 × 10 ⁵/ mL cell suspension, be inoculated in culturing bottle, select low sugar DMEM or RPMI 1640 substratum, wherein containing 20mL/L foetal calf serum, 10nmol/L Regular Insulin, after cultivating 48h, make Hemapoiesis individual layer, cellular form is fusiformis, there is not yet or rarely seenly be less than 10% little circular cell, attached cell converges rate and reaches 55% ~ 65%, be in when will enter or just enter logarithmic phase, adopt the method for liposome transfection, by recon SV40T/pcDNA3.1 transfection in inborn defect cell, or by SV40 direct infection in inborn defect cell, with the liquid medium-selection 1wk containing 700mg/L G418, change G418 concentration into 300mg/L, to occurring positive colony, transfer them to new culturing bottle to expand, Secondary Culture, method 3: above-mentioned cell suspension is made into 5 × 10 with low sugar DMEM or RPMI RPMI-1640 ⁸the cell suspension inoculation of/L final concentration in 24 well culture plates, until cell grow to about 90% merge time for transfection, by the recon SV40T/pcDNA3.1 of 0.2 μ g content, adjusting volume with DMEM or RPMI 1640 is 50 μ l, room temperature place 5min, liposome 6 μ l, adjusting volume with DMEM is 50 μ l, room temperature places 5min, two kinds of reagent mix gently, room temperature places 20min, cell DMEM in 24 orifice plates is washed 3 times simultaneously, every hole adds nutrient solution 100 μ l, adds liposome-SV40T/pcDNA3.1 mixed solution, gently cell surface paving evenly, 6h is placed in 37 DEG C of CO2 incubators, use 0.01g/L collagenase by cell dissociation subsequently, proceed in 6 orifice plates, add perfect medium, adding G418 microbiotic next day makes final concentration be 500mg/L, until there is monoclonal cell to grow.There is monoclonal cell colony to grow after about 10d, choose to be placed in 24 orifice plates and continue to cultivate, maintain and stabilize with the G418 of 300mg/L the amplification cultivation that goes down to posterity.

5, the going down to posterity of inborn defect clone, increase: collect the above-mentioned positive monoclonal cell colony importing SV40 large T antigen gene through lipofection, be made into about 1 × 10 with low sugar DMEM or RPMI 1640 substratum ⁵the cell suspension of/mL, is inoculated in several bottles of 20 ~ 50cm ²in culturing bottle, add 5mL containing 20mL/L foetal calf serum, the low sugar DMEM of 10nmol/L Regular Insulin or RPMI 1640 substratum, to cell attachment growth, converge rate reach 80 ~ 85%, be in the early stage of logarithmic phase time collecting cell, again by above-mentioned steps Secondary Culture, inoculation of repeatedly going down to posterity like this, cultivation, record algebraically and observation of cell growth characteristic.

6, the authentication method of inborn defect clone: after using SV40 to set up clone, the key issue of qualification has: is that this cell of requirement has lasting multiplication capacity, i.e. T antigen stably express in clone; Two are its forms of requirement, basic physiological function isophenous remains unchanged.1. morphological observation: under inverted light microscope every day observation of cell whether in typical epithelial cell sample adherent growth, whether cell proliferation rate very fast, whether in immortalized cell line, typical " S " feature or " arched roof " are formed growth characteristics; With the Potamogeton crispus of transmission electron microscope observing cell, comprise whether entoblast increases, in born of the same parents whether containing abundant glycogenosome.2. Immunohistochemical detection: the visible a large amount of brown particle of dyeing in the nucleus of SV40 transfection, shows that SV40T antigen has been integrated in cell.3. the detection of SV40T antigen: utilize RT-PCR to detect the expression of T antigen in cell, the wherein primer of T antigen: upstream primer (A4239) 5 '-GTT ATG ATT ATAACT GTT ATG-3 ', downstream primer (S4496) 5 '-GAA ATG CCA TCT AGT GAT-3 '; Amplified production length is 268bp, and amplification condition is 94 DEG C, 5min, that is: and (94 DEG C, 1min; 55 DEG C, 1min ,-0.5 DEG C/circulation; 72 DEG C, 1min) × 30, (94 DEG C, 30S; 40 DEG C, 30S, 72 DEG C, 30S) × 15, amplification system is 50 μ l:[Mg ²⁺] 2mmol/L, dNTPs 200 μm of ol/L, primer concentration 0.4 μm of ol/L, Taq 1U, template 5 μ l; Experimental group with the 19th generation cell cDNA for template (with reference to commercially available cDNA first chain synthetic agent box carry out cDNA first chain synthesis, product-20 DEG C preservation); Negative control establishes two, template is done respectively with the cDNA of sterilized water, primary cell, positive control is that template (extracts SV40DNA with reference to SDS-proteinase-K pathway with SV40DNA, because SV40 virus is without coating, do not use SDS rupture of membranes, get 5 μ l and carry out 1.5% agarose gel electrophoresis detection, all the other-20 DEG C save backup); T antigen mRNA RT-PCR product checks order: the amplified production getting 100 μ l systems, reclaim test kit (Takara, Japan) with gel and reclaim product, get 2 μ l DNA solutions and dilute 100 times, survey concentration, remaining DNA and each 10 μ l of upstream and downstream primer checks order.4. Flow cytometry: the cell proportion detecting synthesis in the 19th continuous cell line, division, if its multiplication capacity obviously strengthens than not building the normal cell being, explanation is the result that SV40 large T antigen is integrated, expressed.5. identify with restriction enzyme: carry out enzyme with restriction enzyme BamH I and cut, 10g/L agarose gel electrophoresis is identified, obtain meet SV40T fragment in GenBank, size is about the band of 2600bp.6. the eliminating of malignant proliferation: identified by chromosome karyotype analysis, if karyotype consists of diploid " 46; XX " or " 46; XY ", or be that the caryogram of cell is identical with not building, then illustrate that vicious transformation (whether occur abnormal DNA colony in available flow cytometry analysis clone simultaneously, if do not had, illustrate that knurl feature does not appear in clone yet) does not occur this clone.Chromosome karyotype analysis method is: reach 85-95% (its medium and small circular cell accounts for 10%) at cell density, to be in the culture of logarithmic phase in by 5mL nutrient solution the 250ug/ml colchicine 100ul adding preheating, mix rearmounted 37 DEG C of incubators 4 hours, blot nutrient solution, add the 2-3mL EDTA-trysinization liquid of preheating, 37 DEG C act on 5 minutes, stop digestion, wash lower attached cell, through centrifugal, hypotonic, fixing, film-making, G aobvious band post analysis karyotype.

7, the store method of inborn defect clone: get the attached cell that growth conditions is good, be in the different generations of logarithmic phase, through digestion, stop and centrifugation (1200r/min, 6min), with the frozen storing liquid 0.5 ~ 1ml re-suspended cell containing methyl-sulphoxide, cell density is 5 × 10 ⁵individual/ml, adds cryopreservation tube, through 4 DEG C, and 0.5h;-20 DEG C, 2h;-70 DEG C, spend the night, enter-196 DEG C of Liquid nitrogen storage.

8, birth defect cell bank (Birth Defect Cell Bank, BDCB): with the histocyte of different times, each area, various birth defect disease kind, be built into inborn defect clone by the present invention, and concentrate preservation.Birth defect cell bank (Birth Defect Cell Bank) also can be got its first English alphabet respectively and be referred to as " BDCB "; Various concrete inborn defect clone adds concrete sick first English alphabet of planting with first English alphabet (BD) of inborn defect (Birth Defect) and is referred to as, and makes numbering, indicates the data information of confession cell person.Its inborn defect clone can retain original biological characteristics and energy permanence, stable performance, concentrate the genetic resources saving inborn defect, research for all kinds of inborn defect provides reliable scientific research material, for inborn defect intervention and accelerate the research of its diagnostic method, improvement and bring new ideas lays the foundation.

Claims (1)

1., for a construction process for the birth defect cell bank of medical field, it is characterized in that:

(1) SV40T/pcDNA3.1 recon is built with BamH I, SV40 large T antigen gene, T4DNA ligase enzyme and genophore pcDNA3.1, through the amplification of competence intestinal bacteria, purifying, with lipofection, recon is imported inborn defect cell, and DNA integrates with it, obtain the inborn defect cell containing positive recombinant with G418 screening;

(2) by inborn defect cell and SV40 or Epstein-Barr virus co-cultivation, obtain and infected viral DNA and the inborn defect cell integrated occurs;

(3) by containing positive recombinant inborn defect cell and infected viral DNA and the inborn defect cell occurring to integrate goes down to posterity, amplification cultivation, build deficient cells system of being born also frozen in-196 DEG C of liquid nitrogen, frozen program is 4 DEG C, 0.5h;-20 DEG C, 2h;-70 DEG C, spend the night, with concentrated preservation inborn defect viable cell resource, solve its scientific research material poor and without the problem of single-minded birth defect cell bank;

Described inborn defect cell refers to lymphocyte, the fetus amniotic fluid cast-off cells of surplus in birth defect disease diagnosis, carry out increasing in the RPMI1640 containing 20% foetal calf serum, 5 ~ 10nmol/L Regular Insulin or the low sugar DMEM nutrient solution containing 20mL/L foetal calf serum, 5 ~ 10nmol/L Regular Insulin, after Secondary Culture, digest with 0.01% collagenase II and obtain;

Import the inborn defect cell of recon in order to lipofection as Secondary Culture, in primary amplification cultivation, its collect index be cell attachment cultivate 3 ~ 4 days, cellular form is fusiformis, small round cell less than 10%, the rate of converging of attached cell reaches 75% ~ 85%, is in collecting cell when just entering logarithmic phase; Import the inborn defect histocyte that goes down to posterity of recon as lipofection, the index of its choose opportunities is cellular form be fusiformis, there is not yet or be rarely seenly less than 10% small round cell, the rate of converging of attached cell reaches 55% ~ 65%, is in culturing cell when entering or just entered logarithmic phase; The index that inborn defect clone is collected be select cell attachment growth converge that rate reaches 80 ~ 85%, collecting cell when being in the early stage of logarithmic phase.